Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids

Previous work has shown that molecular phylogenies of plastids, cyanobacteria, and proteobacteria based on the rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) genes rbcL and rbcS are incongruent with molecular phylogenies based on other genes and are also incompatible with structural and biochemical information. Although it has been much speculated that this is the consequence of a single horizontal gene transfer (of a proteobacterial or mitochondrial rubisco operon into plastids of rhodophytic and chromophytic algae), neither this hypothesis nor the alternative hypothesis of ancient gene duplication have been examined in detail. We have conducted phylogenetic analyses of all available bacterial rbcL sequences, and representative plastid sequences, in order to explore these alternative hypothesis and fully examine the complexity of rubisco gene evolution. The rbcL phylogeny reveals a surprising number of gene relationships that are fundamentally incongruent with organismal relationships as inferred from multiple lines of other molecular evidence. On the order of six horizontal gene transfers are implied by the form I (L8S8) rbcL phylogeny, two between cyanobacteria and proteobacteria, one between proteobacteria and plastids, and three within proteobacteria. Alternatively, a single ancient duplication of the form I rubisco operon, followed by repeated and pervasive differential loss of one operon or the other, would account for much of this incongruity. In all probability, the rubisco operon has undergone multiple events of both horizontal gene transfer and gene duplication in different lineages.      

Application of Palladium-Mediated 18F-Fluorination to PET Radiotracer Development: Overcoming Hurdles to Translation

New chemistry methods for the synthesis of radiolabeled small molecules have the potential to impact clinical positron emission tomography (PET) imaging, if they can be successfully translated. However, progression of modern reactions from the stage of synthetic chemistry development to the preparation of radiotracer doses ready for use in human PET imaging is challenging and rare. Here we describe the process of and the successful translation of a modern palladium-mediated fluorination reaction to non-human primate (NHP) baboon PET imaging–an important milestone on the path to human PET imaging. The method, which transforms [¹⁸F]fluoride into an electrophilic fluorination reagent, provides access to aryl–¹⁸F bonds that would be challenging to synthesize via conventional radiochemistry methods.     

High‐throughput screening techniques for rapid PEG‐based precipitation of IgG4 mAb from clarified cell culture supernatant

Locating optimal protein precipitation conditions for complex biological feed materials is problematic. This article describes the application of a series of high‐throughput platforms for the rapid identification and selection of conditions for the precipitation of an IgG₄ monoclonal antibody (mAb) from a complex feedstock using only microliter quantities of material. The approach uses 96‐microwell filter plates combined with high‐throughput analytical methods and a method for well volume determination for product quantification. The low material, time and resource requirements facilitated the use of a full factorial Design of Experiments (DoE) for the rapid investigation into how critical parameters impact the IgG₄ precipitation. To aid the DoE, a set of preliminary range‐finding studies were conducted first. Data collected through this approach describing Polyethylene Glycol (PEG) precipitation of the IgG₄ as a function of mAb concentration, precipitant concentration, and pH are presented. Response surface diagrams were used to explore interactions between parameters and to inform selection of the most favorable conditions for maximum yield and purification. PEG concentrations required for maximum yield and purity were dependant on the IgG₄ concentration; however, concentrations of 14 to 20% w/v, pH 6.5, gave optimal levels of yield and purity. Application of the high‐throughput approach enabled 1,155 conditions to be examined with less than 1 g of material. The level of insights gained over such a short time frame is indicative of the power of microwell experimentation in allowing the rapid identification of appropriate processing conditions for key bioprocess operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Extremely low priming doses of X radiation induce an adaptive response for chromosomal inversions in pKZ1 mouse prostate

An adaptive response is a response to a stress such as radiation exposure that results in a lower than expected biological response. We describe an adaptive response to X radiation in mouse prostate using the pKZ1 chromosomal inversion assay. pKZ1 mice were treated with a priming dose of 0.001, 0.01, 1 or 10 mGy followed 4 h later by a 1000-mGy challenge dose. All priming doses caused a similar reduction in inversions compared to the 1000-mGy group, supporting the hypothesis that the adaptive response is the result of an on/off mechanism. The adaptive response was induced by a priming dose of 0.001 mGy, which is three orders of magnitude lower than has been reported previously. The adaptive responses completely protected against the inversions that would have been induced by a single 1000-mGy dose as well as against a proportion of spontaneous background inversions. The distribution of inversions across prostate gland cross sections after priming plus challenge irradiation suggested that adaptive responses were predominantly due to reduced low-dose radiation-induced inversions rather than to reduced high-dose radiation-induced inversions. This study used radiation doses relevant to human exposure.     

Listeria monocytogenes at the human–wildlife interface: black bears (Ursus americanus) as potential vehicles for Listeria

Listeria monocytogenes is the causative agent of the foodborne illness listeriosis, which can result in severe symptoms and death in susceptible humans and other animals. L. monocytogenes is ubiquitous in the environment and isolates from food and food processing, and clinical sources have been extensively characterized. However, limited information is available on L. monocytogenes from wildlife, especially from urban or suburban settings. As urban and suburban areas are expanding worldwide, humans are increasingly encroaching into wildlife habitats, enhancing the frequency of human–wildlife contacts and associated pathogen transfer events. We investigated the prevalence and characteristics of L. monocytogenes in 231 wild black bear capture events between 2014 and 2017 in urban and suburban sites in North Carolina, Georgia, Virginia and United States, with samples derived from 183 different bears. Of the 231 captures, 105 (45%) yielded L. monocytogenes either alone or together with other Listeria. Analysis of 501 samples, primarily faeces, rectal and nasal swabs for Listeria spp., yielded 777 isolates, of which 537 (70%) were L. monocytogenes. Most L. monocytogenes isolates exhibited serotypes commonly associated with human disease: serotype 1/2a or 3a (57%), followed by the serotype 4b complex (33%). Interestingly, approximately 50% of the serotype 4b isolates had the IVb-v1 profile, associated with emerging clones of L. monocytogenes. Thus, black bears may serve as novel vehicles for L. monocytogenes, including potentially emerging clones. Our results have significant public health implications as they suggest that the ursine host may preferentially select for L. monocytogenes of clinically relevant lineages over the diverse listerial populations in the environment. These findings also help to elucidate the ecology of L. monocytogenes and highlight the public health significance of the human–wildlife interface.